The present invention is directed to monoclonal antibodies capable of specifically recognizing an antigenic determinant (epitope) of the protein kaposin or a derivative thereof and capable of binding thereto, hybridoma cell lines producing said monoclonal antibodies, diagnostic systems for the detection of the presence of a kaposin protein or a derivative thereof as well as antibodies directed against kaposin protein or a derivative thereof, methods for detection of the expression of kaposin protein or a derivative thereof in a biological sample, methods for the detection of antibodies directed against kaposin protein or a derivative thereof, uses of the monoclonal antibodies provided according to the invention and uses of kaposin protein or a derivative thereof, each in diagnostics and therapy.
The human herpes virus 8 has been detected in all forms of Kaposi""s sarcoma, in primary effusion lymphomas (PEL), in Castleman""s disease, in angiosarcomas, in skin lesions of patients who underwent transplantations, in plasmacytomas, sarcoidosis as well as in healthy control individuals (Chang et al., 1994; Boshoff and Weiss, 1997). Seroepidemiological studies have shown that in northern and central Europe HHV-8 is substantially restricted to risk groups and that there are large differences with respect to geography and age.
Moreover, these studies were able to show that in patients with Kaposi""s sarcomas a seroconversion for HHV-8 is detectable months to years prior to the diagnosis of Kaposi""s sarcoma and that it is likely that HHV-8 is transferred primarily via sexual contacts. To date, the clinical signs of a primary HHV-8 infection are unknown. Due to the detection of HHV-8 in almost 100% of all Kaposi""s sarcomas, the correlation of the regional seroprevalence with the incidence of HHV-8 and the seroconversion prior to the appearence of Kaposi""s sarcoma it is believed today that HHV-8 at least represents a co-factor in the tumorigenesis of KS. Up to now, the role of HHV-8 in the other diseases mentioned above is unclear and the question whether HHV-8 is involved in other so far unidentified diseases is still unanswered.
Taxonomically, on the basis of sequence homologies HHV-8 belongs to the gamma herpes virus sub-family and is closely related to EBV and Herpesvirus saimiri. The HHV-8 genome is 140 kb in size and is flanked by several repetitive sequences having a length of approximately 800 bp (Russo et al., 1996). HHV-8 codes for about 80 proteins, 10 of which show homology to cellular gene products (Neipel et al., 1997). Similar to all other herpes viruses, HHV-8 is able to cause a lytic infection which then becomes a latent infection. In the latent phase, at least two viral transcripts are expressed: a differentially spliced mRNA encoding the v-cyclin, v-flip and LANA proteins, as well as T0.7, a short RNA 0.7 kb in length and of up to now unknown function (Zhong et al., 1996). The viral transcript T0.7 is the most abundant of the RNAs expressed in the latent phase and has three open reading frames corresponding to 60, 35, and 47 amino acids.
So far, a HHV-8 infection has been detected by polymerase chain reaction using HHV-8-specific oligonucleotide primers. This direct method of detection has disadvantages in that it (i) requires at lot of effort and costs (about five to ten times the price of serological detection), (ii) is susceptible to false positive results due to contamination, (iii) detects only acute infections but not earlier ones, and (iv) detects only 50% of all (acutely) infected subjects if used on peripheral blood.
The serological detection methods developed so far are based on either the use of HHV-8-infected cell lines or on recombinant viral proteins. Assays detecting the antibodies directed against HHV-8 on HHV-8-positive cell lines by means of immunofluorescence have the disadvantages that (i) their reproducibility is low (i.a. because it is impossible to keep the culture conditions for the HHV-8 cell lines absolutely constant), (ii) their evaluation may not be performed by machine which makes them unsuitable for a larger number of tests, and (iii) it is in part difficult to exclude cross-reactions of antibodies against other viruses. A common problem of all of the assays based on recombinant viral proteins ist their low sensitivity. This problem is based on the fact that antibodies are produced only against particular proteins of the virus and that different individuals may in part produce antibodies against different proteins. The viral proteins tested to date for their utility in serological diagnostics have a sensitivity of only between 30 and 80%. FIG. 1 exemplarily shows that antibodies against the minor capsid protein VP23 can be detected in only 30% of KS patients (FIG. 1). It is highly likely that the use of more than one viral protein is required in order to develop more sensitive assays.
Since with respect to its frequency Kaposi""s sarcoma is in third place of the tumors occuring after an organ transplantation and according to current knowledge its occurence is closely associated to HHV-8 infection it may be expected that in the future organ donors and possibly also blood products will be tested for HHV-8 similar to e.g. the obligatory testing for HIV, hepatitis B and C which is performed today.
In part, the dignity of KS is highly variable. The KS which does not occur endemically is almost exclusively restricted to the immunodeficient patient and generally has a malignant course. It is treated by chemotherapy (such as liposomal doxorubicin), surgically or by means of radiotherapy with modest success. Retrospective studies indicate that the virostatics Foscarnet and Gancyclovir are effective. Larger prospective studies with regard to the effectivity of virostatics on KS have not been published to date. Also missing up to now is a uniform treatment schedule for the B cell lymphomas in which HHV-8 has been detected. Chemotherapeutic schedules have been widely used which were generally employed in the treatment of Non-Hodgekin lymphomas.
Therefore, it is an object of the present invention to provide novel means for the diagnostics and therapy of HHV-8 infections and of dieseases directly or indirectly caused by HHV-8.
According to the invention, this object has been achieved by the monoclonal antibodies characterized in claim 1 as well as by the hybridoma cell lines, diagnostic systems, methods of detection, and uses characterized in more detail in the dependent claims. Preferred embodiments of the invention are obvious from the dependent claims.
According to the invention, there are provided monoclonal antibodies specifically recognizing an epitope of the kaposin protein or a derivative thereof and binding to said epitope. An xe2x80x9cepitopexe2x80x9d according to the invention is intended to mean an antigenic determinant of kaposin peptide or protein or a derivative thereof which may be recognized by a monoclonal antibody and to which the monoclonal antibody can bind.
The derivative of kaposin peptide comprises an amino acid sequence of kaposin peptide formed by deletion, substitution, insertion, addition and/or chemical modification of one or more amino acid(s) provided that the antibody directed against an epitope of the derivative of kaposin peptide is useful for the specific detection of a HHV-8 infection.
The monoclonal antibody provided by the invention recognizes an epitope in the cytoplasmic region or the extracellular region of kaposin protein or a derivative thereof.
The monoclonal antibodies used in the present invention are specifically directed against an epitope of one of the following peptides:
(a) AIPPLVCLLA (SEQ ID NO:13) or
(b) QRGPVAFRTRVATG (SEQ ID NO:23).
A further embodiment also comprises the following sequence QRGPVAFRTRVA (SEQ ID NO:24), composed of the peptides 15 and 16 of Table 1.
In another embodiment of the invention the monoclonal antibody is directed against a peptide fragment (a derivative) thereof having a length of at least 5, at least 6, or at least 7 amino acids.
xe2x80x9cKaposin proteinxe2x80x9d according to the invention is intended to mean a protein having the amino acid sequence shown in FIG. 2 (SEQ ID NO:1) or partial sequences of the protein (peptides). Comprised by the invention are also above mentioned peptides and derivatives thereof having an amino acid sequence formed by deletion, substitution, insertion, addition, and/or chemical modification of one or more amino acid(s) provided that the derivative is useful for the specific detection of a HHV-8 infection and/or provided that a monoclonal antibody may be directed against the derivative of kaposin peptide, useful for diagnostics and/or therapy of HHV-8 infections and diseases.
Also, the invention relates to hybridoma cell lines producing a monoclonal antibody as characterized in more detail hereinabove.
According to the invention, also diagnostic systems in the form of a kit are provided. These diagnostic systems are present in two embodiments.
In the first embodiment, the diagnostic system serves for the detection of a kaposin protein or a derivative thereof, as defined above. The diagnostic system contains at least a monoclonal antibody, as defined in more detail above, in a container. Typically, the kit systems comprise the antibody in labeled or unlabeled forms, reagents to perform the necessary incubations, and substrates or derivatizing agents which will be employed depending on the labeling used.
The detection of the presence or the expression, respectively, of kaposin protein or a derivative thereof in a biological sample may be performed for example as follows: The sample is contacted with a monoclonal antibody, as described above, under conditions such that the antibody is able to bind to an antigenic component of the sample; then, the proportion of antibody binding to the antigenic component in the sample is determined.
As the sample, for example a body liquid, an intact cell, a cellular extract, or a tissue will be used. The proportion of binding of the antibody may be for example determined by immunocytochemical or immunohistochemical staining.
In a second embodiment of the invention, the diagnostic system is designed to be capable of detecting monoclonal or polyclonal antibodies present in a sample, such as a body liquid, an intact cell, a cellular extract, or a tissue. For this purpose, a kaposin peptide or a derivative thereof, as defined above and in claim 1, is provided in an appropriate form in a suitable container, the protein or the derivative thereof being capable of undergoing a immunereaction with the monoclonal or polyclonal antibody to be detected. The sample is contacted with the kaposin peptide or a derivative thereof under conditions suitable to enable binding of the antibody present or presumed to be present in the sample directed against the kaposin peptide or a derivative thereof; furthermore, the proportion of binding of the antibody in the sample to the kaposin peptide of the derivative thereof is determined.